Energy for Sustainable Development 27 (2015) 84–92

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Energy for Sustainable Development

Cultivation of Pleurotus spp. on a combination of anaerobically digestedplant material and various agro-residues

H.N. Chanakya ⁎, Sreesha Malayil, C. VijayalakshmiCentre for Sustainable Technologies (CST), Indian Institute of Science, Bangalore 560012, India

⁎ Corresponding author. Tel.: +91 80 22933046; fax: +E-mail address: chanakya@astra.iisc.ernet.in (H.N. Cha

http://dx.doi.org/10.1016/j.esd.2015.04.0070973-0826/© 2015 Published by Elsevier Inc. on behalf of

a b s t r a c t

a r t i c l e i n f o

Article history:Received 22 October 2013Revised 6 February 2014Accepted 27 April 2015Available online xxxx

Keywords:Pleurotus spp.Biogas digester residueMushroom cultivationPaddy strawCoir pithAgro-residue

Two species of Pleurotus, Pleurotus florida and Pleurotus flabellatuswere cultivated on two agro-residues (paddystraw; PS and coir pith; CP) singly as well as in combination with biogas digester residue (BDR, main feed leafbiomass). The biological efficiency, nutritional value, composition and nutrient balance (C, N and P) achievedwith these substrates were studied. The most suitable substrate that produced higher yields and biologicalefficiency was PS mixed with BDR followed by coir pith with BDR. Addition of BDR with agro-residues couldincrease mushroom yield by 20–30%. The biological efficiency achieved was high for PS + BDR (231.93% forP. florida and 209.92% for P. flabellatus) and for CP + BDR (148.31% for P. florida and 188.46% for P. flabellatus).The OC (organic carbon), TKN (nitrogen) and TP (phosphate) removal of the Pleurotus spp. under investigationsuggests that PS with BDR is the best substrate for growing mushroom.

© 2015 Published by Elsevier Inc. on behalf of International Energy Initiative.

Introduction

Pleurotus spp. is the most extensively cultivated mushroom totalinga production of 9 Mt/year. China alone produces 8 Mt/year. Such exten-sive cultivation is because of its flexible range of requirements oftemperature and other environmental conditions (Swaminathan et al.,1995). These as primary wood rot fungi are able to colonize varioustypes of agricultural waste as substrates. Generally Pleurotus spp.are cultivated on single substrates such as paddy or wheat straws.Various groups around the world have tried to utilize other agro-residues such as maize stover, coir pith, ground nut husk, sugarcanebagasse and mixes of these wastes as substrates (Bisaria et al., 1987;Chang et al., 1981; Ragunathan et al., 1996; Ramamoorthy et al., 1999;Ragunathan and Swaminathan, 2003; Sangwan and Saini, 1995).

Production and cultivation of mushroom are major industries inmany countries where raw materials and the preparation of selectivecompost for mushroom production constitute the major cost inputs(Royse and Sanchez, 2008; Van Roestel, 1988; Wuest, 1983). Therefore,growers seek new ways to lower production costs by increasing bio-efficiency, i.e., producing greater mushroom yield from lesser rawmaterials (Royse, 2010). Though India's present share in the worldproduction and trade of oyster mushroom is meager, being only anestimated 2000 t, thepotential for the future is rated as high for a varietyof reasons. Large quantities of various primary substrates such aswheatstraw, paddy straw, bagasse, chicken manure, tea waste, and de-oiled

91 80 23600683.nakya).

International Energy Initiative.

cakes are easily available all over India and can therefore be most suit-able and inexpensive substrates for expanding future cultivation.Agro-residue production in India estimated at around 1200 Mt(550 Mt surplus) out of which paddy straw is 217 Mt and coir pith is6.9 Mt (Chanakya and Malayil, 2012a; ). Even though paddy straw hasa variety of uses such as animal fodder, animal bedding, domestic fuel,raw material for paper and board making or building material, there isstill a surplus in India. Coir pith on the other had has very few uses,mainly used as compost, and can be used as a potential feedstock forcultivation of mushroom. Coir pith is difficult to compost under normalconditions and requires a very long time. During the rainy season, thetannins and phenols of the coir pith are leached out into the soil andinto the irrigation canals, thereby causingwater pollution. Their conver-sion to mushroom has the potential to avoid this environmentalproblem.

Anaerobic digestion of plant biomass in a plug-flow reactor (PFR,Chanakya andMalayil, 2012a, 2012b) gives two by-products— digestedplant material (henceforth called biogas digester residue, BDR) andbiogas digester liquid (BDL) along with biogas — the main output.Both the digester liquid and the digested biomass are rich in nutrientslike N, P, K and trace elements (Chanakya and Malayil, 2013). Thedigested biomass is commonly used as fertilizer and the digester liquidas pest repellent (Rivard et al., 1995; Tiwari et al., 2000; Yamamotoet al., 2006). The anaerobically digested biomass contains 25% moreaccessible ammonium (NH4

+–N) than untreated liquid manure(Monnet, 2003). However, when its is applied directly to soil as fertiliz-er, it leads to leaching into ground water and volatilization of majornutrients like ammonia takes place which will decrease the efficiency

85H.N. Chanakya et al. / Energy for Sustainable Development 27 (2015) 84–92

of nutrient recycling (Möller et al., 2008; Rivard et al., 1995). Mush-rooms such as Pleurotus spp. are a promising possibility to recyclenutrients as they have been used for centuries to produce protein richfood from agro-residues. Spent biomass from biogas plants retainsabout 40% to 60% of the cellulose and higher extent of lignin (fromoriginal feedstock) thus providing adequate nitrogen rich substratesfor the cultivation of edible mushrooms with potential to generatecash outflow along with the use of biogas plants in the rural areas.BDR, by virtue of its high nitrogen content, when applied to soil directlyas fertilizer, leads to rapid nitrification and denitrification of the addedorganic nitrogen. This in turn leads to lower N-efficiency. Further, basid-iomycetes are the only class of organismswhich can feed on the partial-ly digested material from the biogas plant containing high ligninand therefore are organisms of importance in managing efficiency ofN-recycling processes.

Spent mushroom compost (SMC) has been explored for a diversityof potential commercial applications such as amendment for plantgrowth media (Medina et al., 2009; Ribasa et al., 2009) and bioremedi-ation agent (Chiu et al., 2009). The use of SMC as feedstock forbioethanol production has been examined by a few groups around theworld (Kapu et al., 2012; Zhu et al., 2013). Many white-rot fungi grow-ing on agro-residues exhibit high levels of lignin degrading enzymeslike laccase and manganese peroxidase (Mishra and Kumar, 2007).The genera of Pleurotus have the ability to produce extracellularligninolytic enzymes: laccase and two peroxidases: manganese depen-dent peroxidase, versatile peroxidase and aryl-alcohol oxidase whichmodify and degrade lignin (Stajic et al., 2006). This ability of Pleurotushas led to their cultivation on various lignin-rich lignocellulosicmaterials such as saw dust, paper byproducts and many agriculturalresidues (Rodriguez et al., 2004). This preference of Pleurotus in achiev-ing higher lignin degradation (Ganguli and Chanakya, 1994) can also beused to recover hemicellulose and cellulose for other applications suchas bioethanol.

In the context of embedding biogas plants into sustainable energy,rural life and livelihoods, conversion of the BDRs to value added prod-ucts such asmushrooms could be of great importance in thewidespreadacceptance of biogas plants and renewable energy programs of coun-tries like India and China (Chanakya and Malayil, 2012b). The processof using biomass as a substrate for biogas in a plug flow reactor thusprovides three outputs such as i. biogas for cooking, ii. digester liquid(as pest repellent; Chanakya and Malayil, 2012a, 2012b) and iii.digested biomass residue which can be converted to mushroom andmany other value added products. This also leads to efficient resourcerecovery and nutrient recycling (Chanakya and Malayil, 2012b). Biogasresidue as a single substrate for mushroom cultivation has been testedby Ganguli and Chanakya (1994) and found to be poor due to thephysical collapse of mushroom substrate after pretreatment and conse-quent infestation by insects. BDR contains many undesirable microor-ganisms and therefore it needs to be sterilized before spawning. Whenautoclaved this BDR softens, becomes a pulpy mass and its structurecollapses. In this process there is very little air space left and it thusimpedes mycelial growth and fruiting body formation. When deployedas the sole substrate for mushroom cultivation, the 100% biogas residuesubstrate collapsed after a 14 day spawn run. Therefore to avoidcollapsing of biomass bed during spawn run (with BDR) and to providebetter aeration, the BDR needs to be mixed with other agro-residues.When BDR was mixed with paddy straw in the order of 0%, 50%and 100%, it was found that 50% mix gave better yields of 1.25 kgmushroom per kg of substrate (bioefficiency = 248%, Ganguli andChanakya, 1994).

The aim of this research was to study the process requirementsand yield of Pleurotus florida and Pleurotus flabellatus on feed stockslike paddy straw and coir pith and in combinationwith BDR. The degra-dation patterns of these feedstocks were monitored to examine thepotential of P. florida and P. flabellatus to degrade lignin for utilizingthe digested BDR and potentially enhance returns from deploying

modern biogas plants and make available potential feedstocks forbioethanol production.

Materials and methods

Collection of substrates and spawn

The mushroom substrates (PS and CP) for growing P. florida andP. flabellatus were obtained from Hassan District, Karnataka, India.Anaerobically digested biomass waste was recovered from a plug flowtype biogas plant developed by CST-ASTRA, IISc fed with leafy biomassas feedstock (mainly banana leaf, Musa sp.). The primary inoculaof P. flabellatus and P. florida were obtained from Indian Institute ofHorticultural Science (IIHR), Bangalore.

Pretreatment of substrate

The substrates thus collected were dried in an oven at 90 °C for 24 h.Oven dried PS was chopped into 5–7 cm length pieces. The choppedmaterial was filled in gunny bags and soaked in fresh tap water for12 h. Excess water was then allowed to drain off and substrates werepasteurized by dipping in hot water at 75 °C for 30 min. BDR was sundried and solarized for seven days. Further, CP and BDR were steamsterilized in order to prevent disintegration of the material during pas-teurization in hot water (Ganguli and Chanakya, 1994). Later the mois-ture content of these substratemixeswas adjusted to 50% and spawningwas carried out. Anaerobically digested biomass material and freshfeedstock were mixed in a ratio of 3:7— a ratio chosen based on previ-ous studies (Ganguli and Chanakya, 1994) where it was reported that a50% substitution gave high yields (N2.4 kgmushroom/kgdrywt ofmix).The mixtures and single biomass feedstocks were filled in perforatedpolythene bags (Bano et al., 1962) after spawning (@10% overallweight). The cultivation was carried out in a humid chamber coveredwith jute cloth in laboratory conditions. The poly-bag cultures were fre-quently sprayed with water every day till the stage of fruiting body ini-tiation in order to maintain an adequate moisture content and highhumidity.

Sampling of substrates

A composite sample of 50 g was collected from different bags atvarious intervals i.e., before spawning, 10th day after spawning, aftercomplete mycelia colonization, at the first harvest and final harvest.These samples were dried in a hot air oven at 90 ± 2 °C. The driedmaterial was later powdered and samples were stored in paper bagsfor further analysis. These samples were used to determine variousparameters in order to estimate residual nutrient status in substratesand consequent uptake efficiencies.

Physico-chemical analysis

The organic carbon fraction, TKN and TP reduction among substratesat various stages of the process were monitored. Organic carbon (OC)fraction of the sample was estimated by dry ashing (Jackson, 1973).Total Kjeldhal Nitrogen (TKN) and total phosphate (TP) were deter-mined by the method outlined by Tandon (1993) and APHA (1975). Inorder to study the degradation pattern of these feedstocks, estimationsof cellulose, hemicellulose and lignin contents were carried out by asequential extraction procedure outlined by Chesson (1978). Theharvested mushrooms were analyzed for moisture and crude protein.The protein content was obtained by Lowry's method. Total weight ofall the fruiting bodies harvested from the three subsequent pickingswasmeasured and reported as the total yield of mushroom. The biolog-ical efficiency (yield of mushroom per kg dry substrate) was calculatedusing the formula given by Chang (1978). The statistical analysis was

86 H.N. Chanakya et al. / Energy for Sustainable Development 27 (2015) 84–92

carried out using R-Studio (version 2.15.2 (2012-10-26)) and otherworksheet packages.

Results and discussion

Mushroom cultivation was attempted on 2 feedstocks (PS and CPsingly as well as in combination with BDR) carried out in a jute-clothcovered humid chamber under laboratory conditions (21–25 °C).Various groups around the world have tried to grow Pleurotus specieson various agro-wastes like PS, wheat straw, etc. and have reportedmaximum yields to occur with PS (Kalmis and Sargin, 2004; Lianget al., 2009; Ragunathan et al., 1996; Yildiz et al., 2002). The additionof BDR to agro-residues was not only expected to increase the yieldsbut also hasten the process of mushroom production (Ganguli andChanakya, 1994). The growth and production stages of Pleurotus sp.are characterized by defined time required to reach the stages of pin-head formation, fruit body emergence, duration required for the firstharvest, and subsequent harvests, etc.

Pin-head initiation and its rapidity

The pin head formation process of mushroom production wasenhanced by admixture with BDR (in this case digested banana leafbiomass; PS amended with BDR; Figs. 1 and 2). A 3 day advancementin the time required for pin head formation for both the Pleurotusspecies was observed in the case of PS and CP mixed with BDR whencompared to their use as a single substrate. This suggests that theaddition of BDR to agro-residues could hasten the fruiting process ofmushroom production. Many authors report primordia initiation totake place around 24–30 days with single feedstocks like PS, CP, maizestover, sugarcane bagasse, wheat straw, etc. while using variousPleurotus species such as Pleurotus ostreatus, Pleurotus sajor-caju, andPleurotus citrinopileatus (Kalmis and Sargin, 2004; Liang et al., 2009;Ragunathan et al., 1996; Yildiz et al., 2002). Figs. 1 and 2 show thegrowth stages of the two Pleurotus spp. cultivated on PS and CP mixed

a)

c)

Fig. 1. (a) Early stages of mushroom bodies P. flabellatus on PS + BDR. (b) Ready to harvest staP. florida. (d) Ready to harvest mushroom — P. florida grown on CP + BDR.

with BDR. The pin-head formation for P. florida was faster for both thefeedstocks mixed with biogas residue when compared to P. flabellatus.

Time for fruiting body emergence

Fruiting body formation for Pleurotus spp. cultivated on both thesubstrates occurred between 20 and 30 days (Figs. 1 and 2). In thisstudy the fruiting body initiation for PS with both P. florida andP. flabellatus was 21 days which was early when compared to previousstudies (Ganguli and Chanakya, 1994; Kalmis and Sargin, 2004; Lianget al., 2009; Ragunathan et al., 1996; Yildiz et al., 2002). The additionof BDR to PS hastened the fruiting body initiation process by 1–2 daysamong the two Pleurotus spp. studied. A fruiting body initiation timeof 28–30 days for PS and 22–27 days for CP with various Pleurotus spp.has been reported (Ragunathan et al., 1996). In the case of CP thefruiting body formation occurred on 24 days for P. florida and 30 daysfor P. flabellatus. The addition of biogas residue to CP reduced fruitingbody initiation by 3–4 days among the two Pleurotus spp. BDR alone,as reported earlier (Ganguli and Chanakya, 1994) does not serve as agood substrate as a single feedstock and with regard to the time forfruiting body formation the time requirement by both the Pleurotusspp. (29 days for P. florida and 31 days for P. flabellatus, Fig. 2). Fromthe above observations we conclude that with regard to fruiting bodyformation time with PS + BDR, P. florida performed better while inthe case of CP + BDR, P. flabellatus performed better.

The mushroom yields are reported as fresh weight of mushroomharvested in 80 days (3 flushes) per unit fresh weight of feedstock.It is evident from Fig. 2 that PS along with BDR gave the highest mush-room productivity. When comparing the two Pleurotus spp., P. floridagave a higher yield of 2.32 kg/kg substrate. Previous study in this labshowed that P. flabellatus gave a maximum yield of 1.25 kg/kg whengrown on a mixture of PS + BDR (Ganguli and Chanakya, 1994, withweed biomass feedstock derived BDR at 50% substitution). In thecurrent study a better yield was obtained with a 30% substitution of PSwith BDR (2.32 kg/kg by P. florida and 2.09 kg/kg by P. flabellatus).

b)

d)

ge of P. flabellatus grown on PS + BDR. (c) CP + BDR with extensive mycelial network of

0

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PS PS+BDR CP CP+BDR BDR

kg/k

g

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Yield (P. florida)Yeild (P. flabellatus)BE (P. florida)BE (P. flabellatus)

P. florida

PS

PS+BDR

CP

CP+BDR

BDR

Days

Pin head formationFruiting body formationFirst flush

P. flabellatus

0 10 20 30 40 500 10 20 30 40 50

PS

PS+BDR

CP

CP+BDR

BDR

Days

Pin head formationFruiting body formationFirst flush

Fig. 2. Effect of substrate combinations on onset of various growth stages such as time required for pin head, fruiting body formation and first flushwith two species of Pleurotus. The yieldsof mushroom from three harvests and bioefficiency values for different combinations of feedstocks and Pleurotus spp. increased yields and efficiency by supplementing with BDR.

87H.N. Chanakya et al. / Energy for Sustainable Development 27 (2015) 84–92

Therefore a 30% substitution was found be more efficient compared toearlier studies (Ganguli and Chanakya, 1994). Further, it is observedthat in this study, firstly the baseline productivity using single feed-stocks was higher than reported earlier (PS 2.2 kg/kg with P. ostreatus;Yildiz et al., 2002; and for CP 0.279 kg/kg for P. sajor-caju, Pleurotusplatypus 0.327 kg/kg and P. citrinopileatus 0.252 kg/kg for 3 flushes;Ragunathan et al., 1996). Secondly, the addition of BDR significantly en-hanced the mushroom yields when mixed with PS or CP.

In the case of CP as feedstock, when comparing the overall yield ofthe two mushroom species, P. flabellatus produced higher yield com-pared to P. florida and this has not been reported before. In the case ofPS, the addition of BDR gave a 37% increase in yield for P. florida and24% for P. flabellatus. Similarly, for CP, the addition of BDR increasedmushroom yields by 18.2% for P. florida and 18.6% for P. flabellatus.However, BDR used singly was found to be a poor substrate with amushroom yield of 0.16–0.17 kg/kg substrate for both species ofPleurotus. The increased level of compaction leading to low air availabil-ity is believed to be the cause of low mushroom yields (Ganguli andChanakya, 1994).

In the case of PS as feedstock, the biological efficiency of conversionexpressed as the fresh weight of mushroom harvested in 80 days (3flushes) per unit dry weight of the substrate was the highest forPS + BDR (209.92% for P. flabellatus and 231.93% for P. florida). This isthe highest biological efficiency reported so far in literature. In thecase of CP + BDR a high biological efficiency was achieved withP. flabellatus (188.46%) and P. florida showed a biological efficiency of148.31%. Similar biological efficiency for CP has been reported byvarious other authors (Ragunathan et al., 1996). The important conclu-sion is that the biological efficiency could be increased by about 10–30%when single feedstocks are mixed with ca. 30% BDR. Data recordedduring the study showed that single feedstocks supplemented withBDR held higher moisture content. This could lead to rapid mycelialgrowth in supplemented substrates and its impact needs to be investi-gated further.

The varied productivity and bio-efficiency of different substrate com-binations may be attributed to differences in both their physical proper-ties and nutritional composition. The poor growth and low yield ofmushroom in the BDR have been reported due to the collapse of theirbulk, low air movement, high water content potentially leading to anaer-obic conditions, etc. Proper gaseous exchange in themushroom substratepacked in bags is essential to acquire more oxygen and remove respiredgases and potentially harmful volatiles (Ganguli and Chanakya, 1994).

Dry matter loss and efficiency

In the present investigation it was found that, the dry matter loss ofthe substrates is in agreement with the mushroom yield and biologicalefficiency for both the Pleurotus spp. (Figs. 4 and 5). Dry matter loss isdirectly proportional to the carbon loss in the substrate. In the presentstudy, higher mushroom yields and biological efficiencies correspondto higher dry matter loss. The dry matter lost was partly assimilatedinto the mushroom fruit bodies and partly lost into the atmosphere ascarbon dioxide due to mushroom respiration. There was around 51%TS removal by P. florida in PS, 54% in PS + BDR, 40% in CP and 37% forCP + BDR in 40 days. Total TS loss in 40 days by P. flabellatus was 50%for PS and PS + BDR, and 35% for both CP and CP + BDR (Fig. 4). Thisshows that the efficiency of conversion of substrate TS to mushroomweight was higher for P. florida with PS + BDR as the substrate.

A correlation was attempted between the % TS lost and weight ofPleurotus spp. harvested among various substrate combinations. Areasonable trend was observed for P. florida raised on various substratecombinations (Fig. 3). In the case of P. florida, a straight line fit suggesteda good correlation between %TS lost and mushroom yield. For yieldsobtained on coir pith, a straight line trend obtained suggested a 23%TS being compartmentalized for hyphal growth themushroom harvest-ed being proportional to %TS lost. A poor correlation was obtainedbetween mushroom yields and %TS lost for all other combinations ofmushroom species and substrate mixes.

P. florida

y = 0.0304x + 0.1104

R2 = 0.5904

0

0.5

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1.5

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TS lost %

Yie

ld (

kg/k

g)

Coir pith

y = 0.0939x - 1.7094

R2 = 0.6185

0.5

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1.5

2

0 10 20 30 40 50 60 70 0 5 10 15 20 25 30 35

TS lost %

Yie

ld (

kg/k

g)

Fig. 3. Correlation of TS lost (%) with yield for P. florida, P. flabellatus and coir pith.

88 H.N. Chanakya et al. / Energy for Sustainable Development 27 (2015) 84–92

Organic carbon removal and efficiency

The changes of organic carbon in P. florida and P. flabellatus onvarious substrates during stages of the process are shown in Fig. 4 andthe mass balance for the process is shown in Fig. 5. The organic carboncontent of substrates gradually reduced over the period of mushroom

P. florida

0

0.001

0.002

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0.004

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PSPS+BDRCPCP+BDR

P. florida

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P. florida

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OC

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/gT

S

PSPS+BDRCPCP+BDR

0 3 6 10 14 17 20 24 27 30 35 40

0 3 6 10 14 17 20 24 27 30 35 40

0 3 6 10 14 17 20 24 27 30 35 40

Fig. 4. Dry matter, nitrogen, phosphorus and organic carb

growth from the initial to final harvest stage. This is possible due tothe accumulation of C in mushroom biomass as well as by loss due torespiration. Under normal composting processes, the organic C contentof digesting feedstock gradually increases with the extent of decompo-sition due to the accumulation of lignin rich substances in the residue(Kausar et al., 2011). However, in this study there is a gradual decrease

P. flabellatus

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P. flabellatus

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P. flabellatus

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0 3 6 10 14 17 20 24 27 30 35 40

0 3 6 10 14 17 20 24 27 30 35 40Days

OC

mg

/gT

S

PSPS+BDRCPCP+BDR

on removal from various substrates by Pleurotus spp.

Fig. 5. Carbon, nitrogen and phosphorus balance of the system.

89H.N. Chanakya et al. / Energy for Sustainable Development 27 (2015) 84–92

in organic C suggesting a higher level of degradation of lignin comparedto the other material in the feedstock as well as due to the presence ofN10% recalcitrant silica in PS that is expected to further depress themeasured organic carbon content (Kausar et al., 2011).

In the present investigation, there was a drastic reduction of organiccarbon content in PS+ BDR (65% for P. florida and 60% for P. flabellatus)and CP+BDR(57% for P. florida and 50% for P.flabellatus). This indicatedthat the carbon utilization by P. florida was higher and in the case ofsubstrate PS + BDR was found to allow a higher level of organic carbonremoval. The rate of organic carbon removal by P. floridawasmaximal inthe first 20 days with more than 50% carbon removal occurring withboth PS and PS + BDR. This C-removal process was delayed in thecase of CP and CP + BDRwhere it took 27 days to achieve a 50% carbonremoval (P. florida). The corresponding time for 50% C removal forP. flabellatus was 27 days for PS, 20 days for PS + BDR, 35 days for CPand 40 days for CP + BDR (Figs. 4 and 5). The reduction in the organiccarbon contents reflects the rate and extent of microbially induceddegradation. The extent of carbon removed (mineralized) by P. florida(respired + harvested biomass + loss in leachate) is around 68% forPS, 76% for PS + BDR, 60% for CP and 57% for CP + BDR (Fig. 4). Thecarbon fixed by P. florida is higher for PS + BDR which correlates withits high yield (Fig. 2). The carbon mineralized by P. flabellatus on theother hand was 60% for PS, 67% for PS + BDR, 56% for CP and 52% forCP + BDR. This suggests that under conditions of the study, P. floridacould utilize a higher fraction of carbon compared to P. flabellatus.

TKN removal and efficiency

The changes in the TKN of various substrates under study are shownin Fig. 4 and themass balance of the overall process is computed in Fig. 5and summarized in Table 1. The highest increase in nitrogen percentagewas observed in PS (42% to 51%), CP (35% to 42%) with the cultivationof P. florida. Variations of TKN in different supplemented and non-supplemented substrates for P. florida and P. flabellatus were observedand graphically presented in Figs. 4 and 5. P. florida utilized 40% of theinitial TKN from PS, 50% in PS + BDR, 26% in CP and 38% in CP + BDRin 40 days (Figs. 4 and 5). These results appear to implicate the higheryield of mushrooms in BDR supplemented agro-residues to the higherTKN uptake (Figs. 4 and 5).

P. flabellatus showed an N utilization of 42% of initial TKN in PS.Corresponding values for PS + BDR is 48%; 37% in CP and 39% inCP + BDR in 40 days. P. flabellatus did not show much efficiency inCP + BDR which explains the low yield with this substrate when com-pared to PS. The ammonia that remains in spent biomass is usuallytransformed into nitrate and escapes as nitrogen gas via nitrificationand denitrification reactions during the maturation stage of spentmushroom compost (Bisaria et al., 1987; Prabhu Desai and Shethy,1991). As a result, inorganic nitrogen content in the matured compostis usually low in the spent mushroom compost. Nitrogen is convertedto ammonia nitrogen and Beyer and Wilkinson (2002) found a directcorrelation between substrate ammonia content and subsequent

Table 1Effect of C:N ratio of different substrates on the growth of Pleurotus spp.

Agriculturalwaste

Carbon % Nitrogen % C:N ratio

Substrate Spentsubstrate

Substrate Spentsubstrate

Substrate Spentsubstrates

P. floridaPS 48.17 31.10 0.42 0.51 114.69 60.98PS + BDR 51.79 27.02 0.68 0.73 76.16 37.01CP 27.41 22.12 0.35 0.42 78.31 50.29CP + BDR 44.14 28.60 0.63 0.62 70.06 46.13BDR 54.01 42.33 0.79 0.76 68.37 55.70

P. flabellatusPS 48.17 35.20 0.42 0.48 114.69 73.33PS + BDR 51.79 37.83 0.68 0.71 76.16 53.28CP 27.41 20.02 0.35 0.34 78.31 58.88CP + BDR 44.14 33.07 0.63 0.60 70.06 55.12BDR 55.01 43.20 0.86 0.84 63.97 51.43

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growth of mushrooms. At the end of the mushroom cultivation, whenthe substrate was analyzed without removing the mycelia, the contentof Nwas approximately the same as the untreated substrate, suggestingthat nitrogenwas incorporated into themycelialmass,whereas in a fewsubstrates N content increased appreciably. This is due to the ability ofPleurotus species to fix atmospheric nitrogenwhichmight have contrib-uted to this increment (Rangaswamy et al., 1975). Thefinal TKN contentin spent mushroom compost is dependent on the initial nitrogen pres-ent in the feed material as well as the degree of decomposition andthis is observed in our study also.

Effect of C:N ratio

The C:N ratio of the substrate (mixture and single feedstock) beforecultivation and after cultivation of Pleurotus spp. is shown in Table 1.From Fig. 4 it is observed that the pattern of N removed occurs quickerthan C removalwherein a fall in N levels occurs till 14 dayswhile a fall inC levels occurs till 17–20 days and then gradually the C and N increase.The addition of BDR to the feedstocks narrowed the C:N ratio consider-ably. In the case of PS, the addition of BDR narrowed the C:N ratio by 44%and in the case of CP the addition of BDR narrowed the C:N ratio by 11%suggesting an increased nitrogen for the growth of P. florida andP. flabellatus. P. florida utilized 54 carbon for every nitrogen and additionof BDR to PS, this was reduced to 39 carbon for every nitrogen utilized(Table 1). For P. flabellatus uptake of C is 41 for every unit of N and inthe case of the admixture with BDR this was reduced to 23. In the caseof CP, P. florida utilized 44 carbon for one nitrogen and in the admixtureit was narrowed to 24. P. flabellatus with CP as substrate utilized 20:1(C:N) and the addition of BDR reduced this ratio to 15:1 (Table 2).

Table 2Moisture and protein content of Pleurotus spp. on various substrates.

Substrates Moisture (%) SD Crude protein (%) SD

P. floridaPS 89.64 0.10 22.50 0.08PS + BDR 90.40 0.06 23.67 0.08CP 88.42 1.21 20.25 0.10CP + BDR 88.97 0.48 21.33 0.17BDR 92.22 0.13 21.97 0.35

P. flabellatusPS 91.88 0.63 19.56 0.64PS + BDR 91.55 0.46 20.87 0.01CP 90.89 0.01 19.12 0.05CP + BDR 91.21 0.52 19.73 0.02BDR 91.22 0.03 20.16 0.03

The changes in the C:N ratio showed that the C:N ratios for allsupplemented and non-supplemented substrates narrowed rapidly.One of the main problems with organic residues with wider C:N ratiois that it will lead to immobilization of N and slow down microbialdecomposition of organic matter. With an increase in the N componentof the substrate, a proportionate decrease in carbon was observed.These results are in accordance with those reported by Shetty andMoorthy (1981), Thilahavathy et al. (1991) and Theradimani andMarimuthu (1991). They reported that the C:N ratio of CP could benarrowed down by cultivation of mushroom. Jadhav et al. (1998) alsoreported that the concentration of nitrogen in spent substrates in-creased with narrowing in the C:N ratio and thus showed possibilityof using spent mushroom as a quality cattle feed.

Nitrogen conservation/temporary immobilization

It has been established that both mycelial growth and fruiting bodydevelopment depend on lignocellulosic materials particularly withreference to their C:N ratio (Theradimani and Marimuthu, 1991; Banikand Nandi 2004). The N content in most of the substrates employedfor mushroom cultivation ranges between 0.5 and 0.8% (C:N = 50–60:1), hence the addition of nitrogen to the substrate helps in gettinghigher mushroom yield (Theradimani and Marimuthu, 1991; Banikand Nandi 2004). Most of the primary colonizers/decomposing micro-organisms cannot utilize the N in BDR as the N in BDR is degraded an-aerobically in a biogas plant. Thus much of the N is utilized in theproduction of fungal biomass and is usually efficient. Basidiomycetesare among the few micro-organisms that can effectively convert this Npresent in BDR to protein which is edible (Leisola et al., 2012;Rodriguez et al., 2004). From Fig. 5 it is seen that supplementing agro-residues with BDR increased the nitrogen content of the substrate andtherefore availability of N for the Pleurotus spp. under study. When theprocess of mushroom cultivation is compared to aerobic composting itcan be seen that there is a higher level of better conservation of N inmushroom cultivation (Fig. 5). If agro-residue (PS) is composted theloss of nitrogen through nitrification, denitrification and as leachatewould account for 66% of the initial nitrogen present. In Table 1 it maybe observed that supplementing the agro-residues with BDR narrowedthe C:N ratio which suggests higher availability of N. The nitrogencontent of the agro-residue + BDR had increased N in the order of0.2% for both P. florida and P. flabellatus. In the case of PS, P. floridaused 68% of the initial carbon and P. flabellatus used 60%. The utilizationof carbon by P. florida from PS + BDR was 76% and by P. flabellatuswas67%. This suggests that the utilization of carbon from the substrate byP. flabellatus was lower than P. florida. This pattern was repeated withCP and CP + BDR whereas the carbon uptake by P. florida andP. flabellatuswas similar when the feedstock was BDR. It may thereforebe concluded that conversion of BDR to mushroom is the most efficientbiological route for i. recovery of N resource present in BDR, ii. bringingit back to the food chainwithout sacrificing the compost potential of thedigested feedstock and iii. creating livelihoods around biogas plants andmaking their use economically attractive.

TP removal and efficiency

The TP removal among the two feedstocks and by Pleurotus spp. isreported in Figs. 4 and 5. The TP removal by P. florida in 40 days for PSwas 45%, PS + BDR is 46%, CP is 28% and CP + BDR is 23%. In the caseof P. flabellatus the TP removal within 40 days for PS is 38%, PS + BDRis 30%, CP is 25% and CP + BDR is 12.5%. The removal efficiency byP. florida was higher in PS whereas in other feedstocks it seems to belesser. In P. flabellatus the removal efficiency for feedstocks emendedwith BDR was lower. However this lower removal efficiency of TP hasnot affected the total yield and this needs further investigation.

Cellulose

0.04

0.08

0.12

0.16

Days

gPS (P. florida)PS+BDR(P. florida)CP(P.florida)CP+BDR(P. florida)PS(P. flabellatus)PS+BDR(P.flabellatus)CP(P. flabellatus)CP+BDR(P. flabellatus)

Hemicellulose

0.04

0.09

0.14

0.19

0.24

Days

g

Lignin

0.04

0.09

0.14

0.19

0.24

0.29

0.34

0.39

0.44

0 10 20 30 40 0 10 20 30 40

0 10 20 30 40

Days

g

Fig. 6. Decomposition pattern of the feedstocks with the Pleurotus spp.

91H.N. Chanakya et al. / Energy for Sustainable Development 27 (2015) 84–92

Decomposition pattern of feedstock

The decomposition pattern of lignin, cellulose and hemicelluloseby P. florida and P. flabellatus on various supplemented and non-supplemented substrates is presented in Fig. 6. Degradation of lignin,cellulose and hemicellulose was noticed from an early stage both inP. florida and P. flabellatus (Fig. 6). The extent of cellulose degradationwas in the range of 50–70% of the dry weight in both supplementedand non-supplemented substrates of CP and PS (Fig. 6). The maximumlignin degradation was observed in the case of CP wherein from aninitial value of 43% it fell to 31% (Fig. 6). The general observation hasbeen that the cellulose content decreased as the days of spawningincreased, for P. florida in both the two supplemented and non-supplemented substrates. The decrease in hemicellulose was relativelygreater than cellulose in CP supplementedwith spent biomass implyingthat the fungi utilized a greater extent of hemicellulose than cellulose.The maximum extent of initial lignin content observed (40–45%) wasin CP + BDR and CP as a single feedstock.

P. floridawas more effective in degrading the lignocellulosic con-tent of agricultural residue among both the feedstocks. Basidiomy-cetes have a unique ability of degrading lignin while retaining mostof the hemicellulose and cellulose intact (Henriksson et al., 2000).This property of basidiomycetes may be utilized for bioethanol pro-duction from lingo-cellulosic biomass where the major difficulty isremoving the lignin component without affecting the hemicelluloseand cellulose removal efficiency (Dent and Brown 1978). Ganguliand Chanakya (1994) have reported a similar pattern in degradationwhere the free sugars and pectins increased in substrate after mush-room cultivation while hemicellulose and cellulose were similar andlignin content reduced significantly.

During mushroom cultivation, agricultural residues provide a reser-voir of carbon in the form of cellulose, hemicellulose and lignin, whichis utilized during the growth of spawn and during fructification(Ganguli and Chanakya, 1994). Materials with higher lignin concentra-tions (N15%) lead to slow decomposition and N immobilization. Becauselignin is an aromatic, branched and complex compound, it requires along period to be broken down by conventional soil microorganisms. Lig-nin contributes to the recalcitrance of agro-wastes to decomposition byoccluding the more easily decomposable polysaccharides — celluloseand hemicellulose. Pleurotus spp. are known to produce a wide range ofhydrolytic and oxidative enzymes that enable them to successfullycolonize, degrade and convert many lignocellulosic substrates to theirbiomass (Bano et al., 1993). Such degradation of lignocellulosicmaterials results from the concerted and synergistic action of manyenzymes including endoglucanases, exoglucanases, b-glucosidases,xylanases, laccases and polyphenol oxidases (Buswell et al., 1996;Diaz-Godinez et al., 2008). Previous studies on wood ear cultivationsuggest that the cellulose content of the substrate and enzymeproductionof themushrooms are important in determining the yield of amushroomcrop (Onyango et al., 2011). The variations observed in yield may also beattributed to the complexity of substrates in terms of their cellulose con-tent resulting in a difference in the rate of degradation by the mushroomenzymes. The present study shows a decline in the cellulose, hemicellu-lose and lignin contents of the spent substrates and is within the normalunderstanding of this process (Ganguli and Chanakya, 1994).

Protein content of mushroom

The protein content of harvestedmushrooms is presented in Table 2.The addition of BDR to PS increased the protein content of P. florida

92 H.N. Chanakya et al. / Energy for Sustainable Development 27 (2015) 84–92

marginally (1.17%) when compared to PS as a single feedstock. In thecase of CP + BDR the increase in protein content for P. florida was alsomarginally higher (1.08%) than CP as a single feedstock. Similarly forP. flabellatus the increase in protein content by the addition of BDR toPS was marginally higher (1.31%) and for CP + BDR was 0.61% higherthan CP alone. From the above data it appears that supplementation ofBDR marginally improves the protein content in mushroom whencompared with the use of agricultural residue as single feedstocks.

Conclusion

The present study was carried out to understand the effects ofthe addition of BDR to agro-residues (PS and CP) used as substrate forgrowing mushroom. Laboratory results indicated that the addition ofBDR to agro-residues could enhance the yield of mushroom in therange of 20–30%. The addition of BDR enhances the N and P contentsof the substrate and the uptake of N, P and C by the Pleurotus spp.used. Between the two feedstocks tried, PS with BDR supplement gavehigher yields while among the two mushroom species P. florida gavebetter performance. The highermushroomyields fromBDR supplemen-tation could substantially improve the economics and value addition byuse of biomass based biogas plants and would enhance the spread ofbiogas technology. However, identifying and optimizing the causes ofhigher yields need further investigation.

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